title: Inactivation of the integrin-linked kinase (ILK) in osteoblasts increases mineralization.
abstract: In osteoblasts, Integrin-Linked Kinase (ILK)-dependent phosphorylation of the cJUN transcriptional coactivator, αNAC, induces the nuclear accumulation of the coactivator and potentiates cJUN-dependent transcription. Mutation of the ILK phosphoacceptor site within the αNAC protein leads to cytoplasmic retention of the coactivator and cell-autonomous increases in osteoblastic activity. In order to gain further insight into the ILK-αNAC signaling cascade, we inactivated ILK using RNA knockdown in osteoblastic cells and engineered mice with specific ablation of ILK in osteoblasts. ILK knockdown in MC3T3-E1 osteoblast-like cells reduced phosphorylation of its downstream target glycogen synthase kinase 3β (GSK3β), which led to cytoplasmic retention of αNAC and increased mineralization with augmented expression of the osteoblastic differentiation markers, pro-α1(I) collagen (col1A1), Bone Sialoprotein (Bsp) and Osteocalcin (Ocn). Cultured ILK-deficient primary osteoblasts also showed increased cytoplasmic αNAC levels, and augmented mineralization with higher Runx2, Col1a1 and Bsp expression. Histomorphometric analysis of bones from mutant mice with ILK-deficient osteoblasts (Col1-Cre;Ilk(-/fl)) revealed transient changes, with increased bone volume in newborn animals that was corrected by two weeks of age. Our data suggest that the ILK-αNAC cascade acts to reduce the pace of osteoblast maturation. We propose that in vivo, functional redundancy is able to compensate for the loss of ILK activity, leading to the absence of an obvious phenotype when osteoblast-specific Ilk-deficient mice reach puberty.
authors.affiliation: Research Unit, Shriners Hospital for Children, Montreal, Quebec H3G 1A6, Canada; Department of Human Genetics, McGill University, Montreal, Quebec H3A 2T5, Canada.
authors.forename: Jad
authors.initials: J
authors.lastname: El-Hoss
authors.forename: Alice
authors.initials: A
authors.lastname: Arabian
authors.forename: Shoukat
authors.initials: S
authors.lastname: Dedhar
authors.forename: René
authors.initials: R
authors.lastname: St-Arnaud
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002113:Calcification, Physiologic; D055785:Gene Knockdown Techniques; D051379:Mice; D010006:Osteoblasts / Q000201:enzymology*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors*; D012313:RNA / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000099200:Scaffold Protein ILK
chemical_list: D017346:Protein Serine-Threonine Kinases; D012313:RNA; D000099200:Scaffold Protein ILK
keywords: 4′,6-diamidino-2-phenylindole; BV/TV; Bone Sialoprotein; Bsp; Ctx; DAPI; ECM; GSK3β; ILK; Integrin-Linked Kinase; Integrins; Ocn; Osteoblast; Osteocalcin; RT-qPCR; alphaNAC; bone volume/tissue volume; col1A1; collagen degradation products; extracellular matrix; glycogen synthase kinase 3β; microcomputed tomography; nascent polypeptide associated complex and coregulator alpha; pro-α1(I) collagen; reverse transcription-quantitative polymerase chain reaction; αNAC; μCT